Treatment of protein aggregation myopathic and neurodegenerative diseases by parenteral administration of trehalose

ABSTRACT

Disclosed is a method of treatment of a disease associated with abnormal protein aggregation comprising parenterally administering pharmaceutical formulations comprising trehalose. Also disclosed is an injectable aqueous pharmaceutical formulation comprising a therapeutically effective amount of trehalose.

This application is a continuation of U.S. patent application Ser. No.14/889,727, filed on Nov. 6, 2015, which is a National Stage Entry ofPCT Application No. PCT/IL2014/050411, filed on May 7, 2014, whichclaims the benefit of priority to U.S. Provisional Application No.61/820,278, filed on May 7, 2013.

TECHNOLOGICAL FIELD

The presently disclosed subject matter relates to use of trehalose inthe treatment of myopathies, neurodegenerative disorders or tauopathiesassociated abnormal protein aggregation.

BACKGROUND ART

References considered to be relevant as background to the presentlydisclosed subject matter are listed below:

-   [1] Brunet, G. et al., 1990 Dystrophie musculaire oculo-pharyngée.    Recensement des families françaises et études généalogiques. Rev    Neurol 4:429-434.-   [2] Blumen, S. C. et al., 1997 Epidemiology and inheritance of    oculopharyngeal muscular dystrophy in Israel. Neuromuscul Disord    7:S38-40.-   [3] Becher, M. W. et al., 2001 Occulopharyngeal Muscular Dystrophy    in Hispanics New Mexicans. JAMA. 286(19):2437-40.-   [4] Grewal, R. J. et al., 1999 Mutation Analysis of Oculopharyngeal    Muscular Dystrophy in Hispanic American Families. Arch Neurol    56(11):1378-1381.-   [5] Yamamoto, A. et al., 2000. Reversal of neuropathology and motor    dysfunction in a conditional model of Huntington's disease. Cell    101:57-66.-   [6] J Meyer-Luehmann, M. et al., 2008. Rapid appearance and local    toxicity of amyloid-beta plaques in a mouse model of Alzheimer's    disease. Nature 451:720-724.-   [7] Spires-Jones, T. L. et al., 2009. Passive immunotherapy rapidly    increases structural plasticity in a mouse model of Alzheimer    disease. Neurobiol Dis 33:213-220.-   [8] McClellan, A. J. & Frydman, 2001. Molecular chaperones and the    art of recognizing a lost cause. Nat Cell Biol 3:E51-E53.-   [9] Winklhofer, K. F. et al., 2001. Geldanamycin restores a    defective heat shock response in vivo. J Biol Chem 276:45160-45167.-   [10] Tanaka, M. et al., 2004. Trehalose alleviates    polyglutamine-mediated pathology in a mouse model of Huntington    disease. Nat Med 10:148-154.-   [11] Davies; J. E. et al., 2006. Trehalose reduces aggregate    formation and delays pathology in a transgenic mouse model of    oculopharengeal muscular dystrophy. Hum Mol Genet 15:23-31.-   [12] WHO Food Additives    (http://www.inchem.org/documents/jecfa/jecmono/v46je05.htm).-   [13] Berg, N. O. et al., 1963. Correlation between morphological    alterations and enzyme activities in the mucosa of the small    intestine. Scand Gastroenterol 8:703-712.-   [14] Hore, P. & Messer, M. 1968. Studies on disaccharidase    activities in the small intestine of domestic cats and other    carnivorous mammals. Comp Biochem Physiol 24:717-725.-   [15] http://www.tga.gov.au/pdf/euguide/ich822602006.pdf.-   [16] Langer R 1990. New methods of drug delivery. Science 28;    249:1527-1533.-   [17] Oral Trehalose Therapy to Reverse Arterial Aging in Middle-Aged    and Older Adults. http://clinicaltrials.gov/ct2/show/NCT01575288.

Acknowledgement of the above references herein is not to be inferred asmeaning that these are, in any way relevant to the patentability of thepresently disclosed subject matter.

BACKGROUND

Several groups of diseases resulting from trinucleotide repeat mutationsare known. These diseases are characterized by abnormal stretches ofamino acids in specific proteins encoded by a mutated gene. The mutantprotein aggregates in cells causing typical citotoxic cellular inclusionbodies.

Disorders identified as protein codon reiteration disorders containexpansions of a homopolymeric stretch of amino acids, specificallypolyglutamine (poly Q) or polyalanine (poly A). At least eightneurodegenerative disorders have been associated with polyglutamineexpansions, including Huntington's disease (HD), spinal and bulbarmuscular atrophy (SBMA), dentatortibral and pallidoluysian atrophy(DRPLA), and several forms of spinocerebellar ataxia (SCA). Polyalanineexpansions are associated with several rare and severe congenitalabnormalities, but also with oculopharyngeal muscular dystrophy (OPMD).Additional conditions associated with polyalanine expansions expansionmay include dystrophic disorders.

One such disease is Oculopharyngeal Muscular Dystrophy (OPMD), a rareinherited myopathy characterized by ptosis, severe dysphagia andproximal limb weakness. Its estimated prevalence is 1:100,000 and thelargest clusters reported were in families of French-Canadians origin inCanada and in the US (prevalence 1:1000), Bukhara Jews in Israel(prevalence 1:600) and Hispanics in New Mexico, Arizona Colorado andCalifornia [1-4]. OPMD is inherited, in most cases, as an autosomaldominant trait with complete penetrance. The disease is equallyprevalent among both genders. The gene associated with the diseaseencodes the binding protein nuclear 1 protein (PABPN 1), a nuclearprotein involved in pre-mRNA polyadenylation, transcription regulation,and mRNA nucleocytoplasmic transport. The mutation causing OPMD resultsin production of an abnormal poly (A) PABPN 1.

The disease is most often diagnosed in the fifth-sixth decades of lifeand progresses throughout the patient's life. By the age of 70, themajority of patients suffer from all or some of the following, symptoms:severe dysphagia, ptosis, tongue atrophy and weakness, lower and upperlimb proximal weakness, dysphonia, limitation in upward gaze and facialmuscle weakness. As ptosis becomes more pronounced patients adapt the“astronomer posture” tilting of the head and upward gaze—furtheraggravating the dysphagia. The dysphagia starts with difficulty inswallowing solid food and progresses to liquids as well. As thedysphagia becomes more severe, patients become malnourished, cachectic,dehydrated and suffer from repeated aspiration pneumonia. OPMD does notseem to shorten life expectancy but is associated with severedebilitation and reduced quality of life.

There is no medical treatment or potential cure for OPMD. Currenttherapeutic strategies are confined to surgical interventions aimed atalleviating ptosis. Repeated cricopharyngeal dilatations are frequentlyused to relieve dysphagia. Myotomy of the upper esophageal sphinctermuscles has also been employed. These procedures may provide onlytemporary relief and do not affect the progression of the disease thateventually leads to severe difficulty in swallowing, recurrentaspiration with increasing risk of aspiration pneumonia and severeweight kiss which are the most common causes of mortality in OPMDpatients.

Accordingly, there is an urgent need for compositions and therapeuticmethods for alleviating the signs and symptoms of oculopharyngealmuscular dystrophy.

Although there are controversies regarding the specific role of proteinaggregates in mechanisms underlying cell degeneration, a widely acceptedview is that protein aggregates, the aggregation process and/or earlyoligomeric species are toxic and pivotal to degenerative pathology. Animportant piece of evidence was provided by studies showing theneurodegenerative process can be shut down and even reversed if caughtin an early stage [5]. Furthermore, newer studies have shown thatturnover of many culprit proteins is rapid rather than slow [6, 7] andthat inclusion formation is reversible as well as ensuingneurotransmitter and, most importantly, behavioral abnormalities.

One of the new therapeutic strategies is to enhance the native cellulardefense mechanisms against misfolded and aggregated proteins, forexample using molecular chaperones which facilitate normal folding andrefolding of abnormal conformations back to the native state [8]. Drugssuch as geldanamycin can modulate and enhance chaperone levels [9].Geldanamycin, however, has substantial toxicity and does not penetratewell the blood-brain barrier. It is also possible to stimulateproteasome activity, although this approach might have the danger ofaltering the turnover of molecules normally regulated by proteasomedegradation. In addition, oral administration of the disaccharidetrehalose, a disaccharide known for its protein stabilizing effect, hasbeen shown in HD mouse model to reduce polyglutamine aggregates, improvemotor dysfunction and extend survival [10]. More recently, thesefindings have been reproduced upon oral administration of trehalose in amouse model of OPMD [11].

Trehalose, a glucose disaccharide (α-G-glucopyranosylα-D-glucopyranoside), is found in many plants, fungi, bacteria, insectsand other invertebrates, where it serves as a natural excipient. Due toits unique physical and biochemical properties demonstrated in itsability to sustain and preserve a wide array of biological molecules,trehalose has found its use in several food and cosmetic products andmost notably in therapeutic products. Trehalose is an approvedingredient in all major markets designated as GRAS food ingredient bythe FDA [12].

Currently a clinical study is being conducted in the US which examinesthe effect of large amounts of orally-administered trehalose in theprevention of arterial aging. In this study, the researchers compare theeffect of Trehalose or Maltose or a combination of the two drugs on abio marker associated with arterial aging [17].

GENERAL DESCRIPTION

In one of its aspects the presently disclosed subject matter provides amethod for treating or alleviating a disease associated with abnormalprotein aggregation and/or inclusion bodies formation in myocytes,neurons and other cells or extracellular compartments, or at least onesymptom associated therewith, in a human subject in need thereofcomprising parenterally administering to said subject a therapeuticallyeffective amount of trehalose or a pharmaceutical formulation comprisinga therapeutically effective amount of trehalose.

In the above and other embodiments the disease in accordance with thepresently disclosed subject matter is any one of poly-alanineaggregation disorder, poly-glutamine aggregation disorder and atauopathy, for example any one of oculopharengeal muscular dystrophy(OPMD), spinocerebellar ataxias (SCA), Friedreich's ataxia, spinal andbulbar muscular atrophy (SBMA), Huntington's disease, Parkinson'sdisease, Alzheimer's disease and amyotrophic lateral sclerosis (ALS),dentatorubral-pallidoluysian atrophy (DRPLA), Pick's disease,Corticobasal degeneration (CBD), Progressive supranuclear palsy (PSP)and Frontotemporal dementia and parkinsonism linked to chromosome 17(FTDP-17).

In the above and other embodiments, the pharmaceutical formulation inaccordance with the presently disclosed subject matter is an injectablesolution for parenteral administration, in particular for intravenous,intramuscular and intraperitoneal administration.

In some embodiments, the pharmaceutical formulation in accordance withthe presently disclosed subject matter comprises trehalose as soleactive ingredient, and optionally further comprises at least onepharmaceutically acceptable additive, carrier, excipient or diluent.

In all embodiments the concentration of trehalose in the formulation inaccordance with the presently disclosed subject matter is between about0.1% (w/v) to about 50% (w/v), in particular about 10% (w/v).

In some embodiments the pharmaceutical formulation comprising trehalosein accordance with the presently disclosed subject matter has anosmolality of from about 280 to about 330 mOsm/Kg.

In the above and other embodiments the pharmaceutical formulation inaccordance with the presently disclosed subject matter comprises lessthan 0.74 endotoxin units per ml solution.

The therapeutically effective amount of trehalose in accordance with thepresently disclosed subject matter is from about 1 gram to about 100gram for each single injection and no more than about 1 gram/kg bodyweight of said subject per day.

In other embodiments the therapeutically effective amount of trehaloseor trehalose comprised in said pharmaceutical formulation isadministered once daily at from about 10 mg/kg/day to about 1gram/kg/day of trehalose.

In further embodiments the therapeutically effective amount of trehaloseor trehalose comprised in said pharmaceutical formulation in accordancewith the presently disclosed subject matter is administered at a singledose of 5, 8, 15, 30, 40 or 50 grams of trehalose.

The therapeutically effective amount of trehalose or pharmaceuticalformulation comprising thereof according to the present disclosure maybe administered chronically or periodically.

In the above and other embodiments the therapeutically effective amountof trehalose or pharmaceutical formulation comprising thereof accordingto the present disclosure is administered at a frequency of between oncedaily to once per month.

In further embodiments the therapeutically effective amount of trehaloseor trehalose comprised in said pharmaceutical formulation according tothe present disclosure is administered at a single injectionadministration once a week.

In still further embodiments the therapeutically effective amount oftrehalose or pharmaceutical formulation comprising the same inaccordance with the presently disclosed subject matter is intravenouslyadministered at a single dose of from about 5 to about 35 grams oftrehalose, which may be administered once daily, once every other day,twice a week, once a week, once every two weeks, once every three weeksor once a month.

In the above and other embodiments the pharmaceutical formulation inaccordance with the presently disclosed subject matter is an injectablesolution and wherein the rate of administration is such that the maximumendotoxin level is less than 5 endotoxin units per kilogram of bodyweight per hour.

In some embodiments administration of the therapeutically effectiveamount of trehalose comprised in said pharmaceutical formulation asherein defined, which is adapted for intravenous administration, iscompleted within from about 75 to about 120 minutes, specifically withinless than 90 minutes.

In other embodiments administration in accordance with the presentlydisclosed subject matter comprises a dosing regimen of equal doses, orgradually increasing doses, or gradually decreasing closes of trehaloseor pharmaceutical formulation comprising the same.

In another one of its aspects the presently disclosed subject matterprovides trehalose or a pharmaceutical formulation comprising same, foruse in a method for treating or alleviating a disease associated withabnormal protein aggregation and/or inclusion bodies formation inmyocytes, neurons and other cells or extracellular compartments or atleast one symptom associated therewith, in a human subject in needthereof, said method comprising parenterally administering to saidsubject a therapeutically effective amount of trehalose or apharmaceutical formulation comprising the same.

In a further aspect the presently disclosed subject matter provides anaqueous pharmaceutical formulation comprising a therapeuticallyeffective amount of trehalose as a sole active ingredient, wherein theformulation has a pH about 4.5 to 7.0 and contains less than 0.74endotoxin units per ml and wherein said pharmaceutical formulation isadapted for parenteral administration.

The presently disclosed subject matter further provides a kitcomprising:

-   -   (a) pharmaceutically acceptable trehalose or active derivative        thereof;    -   (b) at least one pharmaceutically acceptable additive, carrier,        excipient and diluent;    -   (c) means for preparing an injectable aqueous solution of the        trehalose by mixing said trehalose with at least one of said        additive, carrier, excipient and diluent;    -   (d) means for parenterally administering said injectable        solution to a patient in need;    -   (e) instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosedherein and to exemplify how it may be carried out in practice,embodiments will now be described, by way of non-limiting example only,with reference to the accompanying drawings, in which:

FIG. 1 Mean plasma concentration of trehalose after i.v. or p.o.administration.

A graph showing the fluctuations in the plasma concentration oftrehalose administered intravenously (iv) or orally (po) to rats, during8 hours post administration.

FIG. 2 Plasma and muscle concentrations trehalose after intravenousadministration

A bar graph showing the fluctuations in plasma and muscle concentrationsof trehalose administered intravenously (1 g/kg) to rats, during 72hours post administration.

DETAILED DESCRIPTION OF EMBODIMENTS

The presently disclosed subject matter is based on the surprisingfinding that upon parenteral, e.g. intravenous administration of anaqueous formulation comprising trehalose, therapeutically effectiveamounts of trehalose reached the plasma and accumulated in muscle of thetested animals as compared to plasma and muscle levels of trehaloseobtained following oral administration of such formulations.

Trehalose is known for its ability to sustain and preserve a wide arrayof biological molecules. Trehalose has been used in a variety ofresearch applications and is contained in several commercially availabletherapeutic products, including Herceptin, Avastin, Lucentis, andAdvate, where it serves mainly as a lyoprotectant. Although trehalose iswidely used as an excipient/additive together with another activeingredient, its use as a therapeutically active ingredient per se israther exceptional.

Trehalose is capable of inhibiting intra-cellular aggregation ofabnormal proteins associated with neurodegenerative diseases andmyopathies. The leading example is the aggregation-suppressing effect oftrehalose on the mutant huntingtin, a polyglutamine protein causingHuntington's disease (HD). Oral administration of trehalose totransgenic HD mice (0.2%-5% trehalose in drinking water consumedspontaneously in the course of 5-9 weeks) led to inhibition of formationof intra-nuclear huntingtin aggregates in the brain and liver and, moreimportantly led to improvement of HD-related motor symptoms.

Interestingly, therapeutic use of trehalose, especially in the contextof neuronal and muscular diseases, is significantly precluded by thefact that trehalose is hydrolyzed by the intrinsic enzyme trehalose atthe epithelial brush border in the small intestine [13, 14], due towhich only a small fraction of any enterally administered dose reachesblood stream, neuronal or muscle-tissues. The present disclosure aims atintroducing a novel therapeutic regime using parenteral administrationof trehalose, thereby achieving higher bioavailability and therapeuticefficacy in the treatment of myopathic and neurodegenerative diseasesassociated with abnormal protein aggregation, specifically polyalaninepolyglutamine and tauopathies, disorders, and OPMD in particular.

Thus the presently disclosed subject matter provides a method fortreatingoOr alleviating a disease associated with abnormal proteinaggregation and/or inclusion bodies formation in myocytes, neurons andother cells or extracellular compartments, or at least one Symptomassociated therewith, in a human subject in need thereof comprisingparenterally administering to said subject, a therapeutically effective,amount of trehalose, or a pharmaceutical formulation comprising atherapeutically effective amount of trehalose.

As herein defined the term “disease associated with abnormal proteinaggregation and/or inclusion bodies formation in myocytes, neurons andother cells or extracellular compartments” refers to any diseaseassociated with protein aggregation or protein misfolding, where thecommon underlying biological feature of these diseases being theaggregation of certain peptides and proteins, thereby generating acascade of pathological events, including the secondary aggregation ofvarious other proteins and the consequent failure of protein homeostasisto preserve normal biological function.

The term “treating or alleviating” as herein defined refers to achievinga therapeutic effect, ameliorating, relieving or reducing the severityand/or frequency of at least one sign or symptom associated withdiseases as herein defined, elimination of signs or symptoms and/orunderlying cause, prevention of the occurrence of symptoms and/or theirunderlying cause (e.g., prophylactic therapy), improvement orremediation of damage and eliminating or reducing the extent of proteinaggregation.

Symptoms associated with the diseases as herein defined include, but arenot limited to, drooping eyelids (a condition known as “ptosis”),difficulty in swallowing (called “dysphagia”), muscle fatigue,movement/motion disorders and cognitive disorders to name but few.

In particular, where the disease to be treated is OPMD, the formulationsand methods described herein are useful in the treatment of the signsand symptoms of OPMD. Signs and symptoms of OPMD include severedysphagia ptosis, tongue atrophy and weakness, lower and upper limbproximal weakness, dysphonia, limitation in upward gaze and facialmuscle weakness.

As known in the art, ample diseases and disorders are defined as“disease associated with abnormal protein aggregation and/or inclusionbodies formation in myocytes, neurons and other cells or extracellularcompartments”. For example, the presently disclosed subject matterprovides a method for treating or alleviating a disease associated withabnormal protein aggregation which is a neurodegenerative disorder, apoly-glutamine or a poly-alanine aggregation disorder, a protein codonreiteration disorder, a myopathy, and a tauopathy, to name but few.

The term “neurodegenerative disorder” as herein defined refers tohereditary or sporadic conditions characterized by progressive nervoussystem dysfunction, specifically disorders of this group that areassociated with formation of abnormal protein aggregates, also known asinclusion bodies formation. Examples for a neurodegenerative disorderwhich is associated with abnormal protein aggregation are Amyotrophiclateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease andothers.

The terms “poly-glutamine”, “poly-alanine aggregation disorder” or“protein codon reiteration disorder” as herein defined refer todisorders associated with formation of intracellular polyglutamine orpolyalanine aggregates, preferably referring to oculopharyngeal musculardystrophy (OPMD), Huntington's disease (HD), spinal and bulbar muscularatrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA) andspinocerebellar ataxia (SCA).

The term “myopathy” as herein defined refers to inherited or acquireddegenerative disease involving atrophy of the muscle fibers, in thecontext of present disclosure particularly referring to OPMD.

The term “tauopathy” as herein defined refers to neurodegenerativediseases associated with tau-pathology, prototypic intracellularaggregation of tau microfilaments, in the context of present disclosureparticularly referring to Pick's disease, Corticobasal degeneration(CBD), progressive supranuclear palsy (PSP) and frontotemporal dementiaand parkinsonism linked to chromosome 17 (FTDP-17).

Tauopathies are known as diseases caused by mutations leading tomisfolding of Tau microtubule-associated protein that binds andstabilizes microtubules in neuronal cells. Tau pathology is a prominentfeature of the sporadic Alzheimer's disease (AD), but is also seen in avariety of other related neurodegenerative diseases, such as Pick'sdisease, Corticobasal degeneration (CBD), Progressive supranuclear palsy(PSP) and Frontotemporal dementia and parkinsonism linked to chromosome17 (FTDP-17). More than 30 different inherited mutations or nucleotidesubstitutions in the FTDP-17 gene on chromosome 17q21 have been relatedto neurodegenerative disease manifesting a prototypic intracellularaggregation of tau microfilaments. Tau mutation and by analogy taudysfunction in inherited and, in sporadic diseases may be pathogenicthrough mechanisms involving both loss of function (decreasedmicrotubules stabilization) and toxic gain of function (increased fibrilformation).

Thus in the above and other embodiments the disease is any one ofpoly-alanine aggregation disorder, poly-glutamine aggregation disorderand a tauopathy.

In the above and other embodiments the disease is as herein defined isany one of oculopharengeal muscular dystrophy (OPMD), spinocerebellarataxias (SCA), Friedreich's ataxia, spinal and bulbar muscular atrophy(SBMA), Huntington's disease, Parkinson's disease, Alzheimer's diseaseand amyotrophic lateral sclerosis (ALS), dentatorubral-pallidoluysianatrophy (DRPLA), Pick's disease, Corticobasal degeneration (CBD),Progressive supranuclear palsy (PSP) and Frontotemporal dementia andparkinsonism linked to chromosome 17 (FTDP-17).

In some embodiments the disease is as herein defined is oculopharengealmuscular dystrophy (OPMD).

As indicated above, OPMD is caused by inherited abnormal expansions ofpolyalanine in the poly A binding protein nuclear 1 protein (PABPN1)leading to PABPN1 misfolding and formation of tubule-filamentous,inclusions/aggregates in nuclei of the affected muscle cells. Oraladministration of trehalose to transgenic OPMD mice (2% trehalose indrinking water consumed spontaneously during 4-6 months) was shown tosignificantly reduce aggregation formation and toxicity of the mutantPABPN1 in myocytes. Overall, oral trehalose was shown to delay the OPMDonset, attenuate the disease phenotype, decrease polyalanine proteinaggregate formation and decrease cell death, thus suggesting thattrehalose may be a potent anti-aggregation therapy for OPMD and otherprotein codon reiteration disorders.

As indicated in the accompanying Examples, the inventors have shown thattrehalose can be successfully delivered to muscle, which is the targetorgan in treatment of various diseases and disorders as herein defined,for example but not limited to OPMD.

Huntington's disease and OPMD share a common genetic basis as both arecaused by inherited, trinucleotide expansion, mutations in a diseasecausative gene. The HD gene (HTT), located on chromosome 4p16, containsrepetitive trinucleotide sequences (CAG)n encoding for a polyglutamine(poly Q) stretch. While the normal HTT gene variants contain 7-35repeats, HD-related variants are above that range and the extent ofrepeat expansions is correlated to the severity and earlier onset of HDsymptoms (the effect termed anticipation). Analogous (CAG)n repeatexpansions mutations were associated with other polyglutamine disorders,such as SBMA, DRPLA and several ataxias belonging to the group of SCA.Similarly, the OPMD-related gene, PABPN1 on chromosome 14q11 containstrinucleotide repeats (CGC)n encoding polyalanine (poly A), wherein(CGC)6 repeats is the normal threshold above which OPMD is anticipatedwith an increased number of repeats.

Thus in other embodiments the disease is as herein defined isHuntington's disease.

In view of the apparent lack of medical therapy for HD, OPMD and otherrelated disorders apart from medications that lessen some motor andpsychiatric symptoms, therapies that directly interfere with thedisease-causing mechanisms are sorely needed.

In some embodiments the disease is as herein defined is spinocerebellarataxia (SCA). As known in the art there are many types ofspinocerebellar ataxia, with the most common Spinocerebellar ataxiaincluding Friedreich's ataxia, SCA 1,3,8, and more. Many SCAs fall underthe category of polyglutamine diseases, which are caused when adisease-associated protein (i.e., ataxin-1, ataxin-3, etc.) contains aglutamine repeat beyond a certain threshold.

In other embodiments disease is as herein defined is spinal and bulbarmuscular atrophy (SBMA). The disease SBMA, also known as spinobulbarmuscular atrophy, bulbo-spinal atrophy, X-linked bulbospinal neuropathy(XBSN), X-linked spinal muscular atrophy type 1 (SMAX1), and Kennedy'sdisease (KD), as known in the art, is a debilitating neurodegenerativedisease resulting in muscle cramps and progressive weakness due todegeneration of motor neurons in the brain stem and spinal cord.

In further embodiments disease is as herein defined is Huntington'sdisease, Parkinson's disease, Alzheimer's disease or amyotrophic lateralsclerosis (ALS).

Huntington's disease (HD) is a neurodegenerative genetic disorder thataffects muscle coordination and leads to cognitive decline andpsychiatric problems; Parkinson's disease is a degenerative disorder ofthe central nervous system; Alzheimer's disease (AD) is manifested byshort term memory loss, confusion, irritability, aggression, moodswings, trouble with language, and long-term memory loss; andamyotrophic lateral sclerosis (ALS, also referred to as motor neuronaldisease or Lou Gehrig's disease) is a neurodegenerative diseasecharacterized by rapidly progressive weakness due to muscle atrophy andmuscle spasticity, difficulty in speaking (dysarthria), swallowing(dysphagia), and breathing (dyspnea).

As indicated above, the presently disclosed subject matter provides amethod for treating or alleviating a disease as herein defined in ahuman subject in need thereof comprising parenterally administering tosaid subject a therapeutically effective amount of trehalose or apharmaceutical formulation comprising same.

In the above reports of trehalose as a therapeutic agent for diseasesassociated with protein aggregation, there is need for exceedingly highand continuous oral intake of trehalose in order to achieve the desiredtherapeutic effect in neuronal or muscle tissue, due to its in theintestines.

The instant disclosure is directed to a therapeutic regimen usingtrehalose that is administered to a subject in need by parenteraladministration.

Thus in some embodiments the pharmaceutical formulation as hereindefined is an injectable solution for parenteral administration.

The term “subject in need thereof” refers to a subject suffering from adisease as herein defined, for example but not limited to HD or OPMD oranother polyglutamine or polyalanine aggregation disorder.

The therapeutically effective amount of trehalose can be from about 10mg to about 1,000 mg/Kg body weight per day. The trehalose can becomprised as active ingredient in a pharmaceutical formulation suitablefor parenteral administration. Administration can be based on a dailyregime as a single dose or multiple doses by a single parenteraladministration or as multiple doses by multiple parenteraladministrations, respectively. Alternatively or additionally,administration can be periodic, for example every other day, three timesweekly, twice weekly, once weekly, or once monthly, and frequency ofadministration can be varied according to the patient condition.

The term parenterally as, herein defined refers to a route ofadministration where the desired effect is systemic and the active agent(herein defined as trehalose), is administered by routes other than thedigestive tract, for example intravenous, intramuscular andintraperitoneal administration.

As indicated above, trehalose is known in the art as a lyoprotectant. Assuch the safety and toxicity of trehalose has been extensivelyinvestigated, and the substance was found to be safe when administeredboth orally and intravenously, in doses that are substantially higherthan the intended therapeutic dose.

As shown in the Examples below, a pharmaceutical formulation comprisingtrehalose as sole active ingredient was parenterally administeredresulting in relatively high plasma and muscle levels.

Thus, in some embodiments the pharmaceutical formulation as hereindefined comprises trehalose as sole active ingredient, and optionallyfurther comprises at least one pharmaceutically acceptable additive,carrier, excipient or diluent.

In other words, in the presently disclosed subject matter trehalose isthe only active agent or ingredient. Notwithstanding the above,trehalose or a pharmaceutically formulation comprising same may be usedin combination with other therapies or treatments for treating oralleviating a disease associated with abnormal protein aggregation.

The term “trehalose” as herein defined refers to a disaccharide glucoseα-G-glucopyranosyl α-D-glucopyranoside. The term trehalose also refersto any active derivative of trehalose, for example hydrides and saltsthereof.

Trehalose is a naturally occurring disaccharide comprised of a 1,1linkage of two D-glucose molecules. It is a non-reducing sugar that isnot easily hydrolyzed by acid. Its molecular formula is C₁₂H₂₂O₁₁ andits molecular weight is 342.31 Dalton. Other-names used to describe theα, α form, the isomer commonly referred to as ‘trehalose’, areα,α-Trehalose, α-δ-glucopyranosyl, α-δ-glucopyranoside, mushroom sugar,mycose.

As indicated above, trehalose is well known for its protein-stabilizingproperties. It is used extensively in many applications as a stabilizerof frozen food, in freeze-drying of biological systems and cells, as astabilizer of therapeutic parenteral proteins and as an excipient intablets and IV solutions. Trehalose is recognized as a GRAS (GenerallyRegarded as Safe) food ingredient by the FDA and is listed on the USP-NF(United States Pharmacopoeia National Formulary), EP (EuropeanPharmacopoeia) and JP (Japanese Pharmacopoeia).

Like all disaccharides, trehalose is metabolized at the epithelial brushborder to two D-glucose molecules. Less than 0.5% of ingested trehaloseis absorbed into the blood stream where it is further metabolized byliver and kidney by trehalose. Oral trehalose in amounts exceeding 40-50gram per day causes diarrhea and bloating. Thus in order to achievetherapeutic amounts of trehalose in the muscle cells it was necessary tocircumvent the massive metabolism in the gastrointestinal tract (GI)tract. Therefore the inventors developed an intravenous (IV) solution oftrehalose.

The term “formulation” as herein defined refers to a compositioncomprising trehalose as active ingredient and optionally furthercomprising additional active ingredient such as anti-inflammatory agent,and at least one pharmaceutically acceptable additive, carrier,excipient or diluents as well known in the art. This formulation mayfurther comprise a trehalose (a glycoside hydrolase enzyme catalyzingthe conversion of trehalose to glucose found in the intestine, kidneyand liver) inhibitor, i.e. competitive or other inhibitor of thetrehalose enzyme.

Particular pharmaceutical formulations are suitable for parenteraladministration, specifically injectable solutions; wherein theconcentration of trehalose in said formulation is between about 0.1%(weight/volume) to about 50% (weight/volume), more specifically whereinthe concentration of trehalose in the formulation as herein defined is,but is not limited to, about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%or 25% (weight/volume).

When referring to trehalose, the term trehalose encompasses not only thespecified molecular entity but also its pharmaceutically acceptable,pharmacologically active analogs, including, but not limited to, salts,polymorphs, esters, amides, prodrugs, adducts, conjugates, activemetabolites, and the like.

Thus the active agent, trehalose, may be administered in the form of thecompound per se as well as in the form of a salt, polymorph, ester,amide, prodrug, derivative, or the like, provided the salt, polymorph,ester, amide, prodrug or derivative is suitable pharmacologically.Salts, esters, amides, prodrugs and other derivatives of the activeagents may be prepared using standard procedures known to those skilledin the art of synthetic organic chemistry and described, for example, byJ. March, Advanced Organic Chemistry: Reactions, Mechanisms andStructure, 4th Ed. (New York: Wiley-Interscience, 1992). For any activeagents that may exist in enantiomeric forms, the active agent may beincorporated into the present formulations either as the racemate or inenantiomerically pure form.

Salts are compounds that ionize in aqueous solutions and may beemployed, for example, to adjust the tonicity of the solution. If theactive agent is present in the forth of a salt, additional salts may beadded to the composition in order, for example, to effect ion exchangewith the active agent. Salts suitable for use with the compositionsdescribed herein are known in the art and include, for example, lithium,sodium, potassium, calcium, and magnesium salts having appropriatecounterions that may be selected from chloride, bromide, iodide,carbonate, phosphate, nitrate, silicate, sulfate, phosphite, nitrite,sulfite, and the like.

Buffers are compounds or solutions that are employed to aid inmaintaining the concentration of an analyte within a desired range. Forexample, pharmaceutically acceptable pH buffers are used to maintain theacidity or basicity of a solution within a pharmaceutically acceptablerange. Buffers for use in the compositions disclosed herein may be anyknown or hereafter discovered buffer.

Excipients are inactive ingredients that may be employed in thecompositions described herein for a variety of reasons. A wide range ofexcipients are described in the literature (e.g., Rowe et al., Handbookof Pharmaceutical Excipients, McGraw Hill, 2006).

Additives and diluents are well known in the art.

In some specific embodiments the pH of the formulation is about 4.5 to7.0; the osmolality of the formulation is about 280-330 mOsm/kg, theformulation contains less than 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, 0.1 or less endotoxin units per mL and the aqueous formulation isabout 50%, 40%, 30%, 20%, 10%, 5% or less trehalose (w/v).

Thus the presently disclosed trehalose delivery systems, for exampleformulations comprising trehalose as sole active ingredient, cangenerally comprise, a buffering agent (pH adjusting additives), an agentwhich adjusts the osmolality thereof, and optionally, one or morepharmaceutically acceptable carriers, excipients and/or additives asknown in the art. Supplementary pharmaceutically acceptable activeingredients can also be incorporated into the formulations.

For formulations as herein defined administered as aqueous or othersolvent-based dosage forms (e.g., for parenteral administration), avariety of liquid carriers may be used, in particular water or saline.Aqueous solutions may include salts, buffers, and the like. The carriercan be solvent or dispersion medium suitable forparenterally-administrable compositions containing, for example, water,saline, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Water is an essential additive (or carrier).

Thus as used herein the term “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Except as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic composition is contemplated. It iscontemplated that the active agent can be delivered by any acceptableparenteral route and in any pharmaceutically acceptable dosage form.

For purposes of parenteral administration, formulations in suitable oilsuch as sesame or peanut oil or in aqueous propylene glycol can beemployed, as well as sterile aqueous solutions of the correspondingwater-soluble salts. Such aqueous formulations may be suitably buffered,if necessary, and the liquid diluent first rendered isotonic withsufficient saline or glucose. These aqueous formulations are especiallysuitable for intravenous, intramuscular, subcutaneous andintraperitoneal injection purposes. In this connection, the sterileaqueous media employed are all readily obtainable by standard techniqueswell-known to those skilled in the art.

Methods of preparing various pharmaceutical formulations with a certainamount of active ingredient are known, or will be apparent in light ofthis disclosure, to those skilled in this art.

As described in the accompanying Examples, the inventors have prepared aspecific injectable, aqueous formulation comprising trehalose, that iscurrently used in an on-going study in OPMD patients, as described belowin Example 6 (and may be used for treatment of other diseases,associated with abnormal protein aggregation).

Given the fact that over 99.5% of the trehalose is not absorbed into theblood stream, and that oral amounts of trehalose higher than 50 g a dayin humans frequently cause diarrhea, bloating and discomfort asindicated above, the most effective way to ensure that adequate amountsof trehalose indeed reach the muscle cells is to circumvent theextensive gut metabolism and administer trehalose by intravenous (IV)solution.

The disclosed injectable solution of trehalose can be intravenouslyadministered to OPMD patients at a single administration (injection)during about 75 to 120 minutes, for example 90 minutes, every day, everyother day, twice a week, once a week, once in 10 days, once every twoweeks or once a month, for a defined number of administrations as willbe determined by the attending physician.

Based on well-known pharmacokinetic profile of trehalose, initial once aWeek, 24-week IV therapy will enable trehalose to enter the muscle cellsand potentially exert its therapeutic effect, previously demonstrated inanimal models. The amounts to be administered in the weekly injectionare based on known safety studies as well as the rate of metabolism andclearance from the blood so as to enable trehalose to reach muscle cellsbefore it is cleared from the blood.

The doses are calculated based on the expected plasma and tissuedistribution of trehalose, effective concentration of trehalose indiseased cells and known safety closes in multiple animal studies.

Following initial treatment, once weekly administration can be chronic,at set time points, for spells of several or scores of weeks, forexample additional consecutive 48 weeks, thus a total of 72 weeks oftreatment, or administration can be periodic.

In some embodiments, the osmolality of the disclosed aqueous trehaloseinjectable pharmaceutical formulation is from about 280 to about 330mOsm/Kg.

As indicated above, the inventors have shown successful delivery oftrehalose to plasma and muscle by parenteral, specifically IVadministration. Thus in some embodiments administration of trehalose ora pharmaceutical formulation comprising same may be in any one ofintravenous, intramuscular and intraperitoneal administration.

In one embodiment the purified trehalose is substantially free ofcontaminants resulting from its enzymatic preparation process, such asorganic solvents used in the process (e.g., ammonium, acetonitrile,acetamide or alcohols), TFA, ether or other contaminants. In thiscontext “substantially” free of contaminants means that the contaminantcontent of any residual peptide originating from the enzymaticpreparation process at the end of the purification process is preferablyless than 0.5%, less than 0.3%, less than 0.25%, less than 0.1%, lessthan 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than0.01%, less than 0.005%, less than 0.003%, or less than 0.001% of thetotal weight of the trehalose. The content of contaminants can bedetermined by conventional methods such as gas chromatography.

In specific embodiments, the residual solvents in the purified trehaloseare less than the limits set in the ICH guidelines, e.g., IMPURITIES:GUIDELINE FOR RESIDUAL SOLVENTS Q3C(R5) (available at:http://www.tga.gov.au/pdf/euguide/ich822602006.pdf) [15]). For example,the purified trehalose contains <5000 ppm ethanol (e.g., <140 ppm),and/or <3000 ppm methanol.

Endotoxins (also known as lipopolysaccharides (LPS) and lipoglycans) arelarge molecules consisting of a lipid and a polysaccharide composed ofO-antigen, outer core and inner core joined by a covalent bond; they arefound in the outer membrane of Gram-negative bacteria, and elicit strongimmune responses in animals. Endotoxins are well-known contaminants insubstances purified by using bacterial systems and their removal is thuscrucial for safety of using therapeutic formulations comprising suchsubstances.

As shown in the accompanying Examples, the presently disclosedformulation comprises less than 5 endotoxin units per kilogram of bodyweight of a patient administered with, the formulation, per hour ofadministration.

Thus in some embodiments the pharmaceutical formulation as herniadefined comprises less than 0.74 endotoxin units per ml solution.

Therapeutically effective amount as herein defined will depend on anumber of factors and will vary from subject to subject and may bedetermined by considerations well known to a skilled person in the fieldof the invention (e.g. a skilled physician). Such factors include theseverity of the symptoms, the patient's age, weight and generalcondition, and the judgment of the prescribing physician.

Generally, by the term “therapeutically effective amount” it is meant anontoxic but sufficient amount of trehalose to provide the desiredeffect, namely the treating or alleviating a disease associated withabnormal protein aggregation and/or inclusion bodies formation inmyocytes, neurons and other cells or extracellular compartments.

The term “therapeutically effective amount or pharmaceutical compositioncomprising thereof” is to be understood as the amount of trehalosecomprised in the administered formulation. The amount of the formulationitself may vary according to the concentration of the trehalosecomprised therein (namely, such that the concentration of trehalose insaid formulation is between about 0.1% (w/v) to about 50% (w/v) and suchthat the maximum endotoxin level in administered formulation is lessthan 5 endotoxin units per kilogram of body weight per hour).

Therapeutically effective amount of trehalose refers to an amount offrom about 1 grain to about 100 gram for each daily injection and nomore than 1 g/kg body weight of said subject per day of a treated humansubject. Amount may considerably vary. Thus the range can be from eachof 10, 20, 50, 75, 100, 150, 200, 300 mg/Kg body weight per day up toeach of 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and1000 mg/Kg body weight per day.

Thus in other embodiments, therapeutically effective amount of trehaloseas herein defined is from about 1 gram to about 100 gram for each singleinjection and no more than about 1 gram/kg body weight of said subjectper day.

The terms “injection” and “infusion” are used interchangeably and referto parenteral administration of the formulation as herein defined.

The therapeutically effective amount of trehalose or pharmaceuticalcomposition comprising thereof is administered parenterally, and can beadministered as a single dose or multiple doses, which may be identicalor different, by a single administration per day, or multiple doses canbe parenterally administered by multiple daily or weekly or monthly ormore prolonged administrations.

A therapeutically effective amount of trehalose or pharmaceuticalcomposition according to the above, administered IV, IP or IM, as asingle dose or multiple doses, which may be identical or different, by asingle administration per day, or optionally as multiple doses, whichmay be identical or different, by multiple administrations per day, orfurther optionally in a dosing regimen of equal doses, or graduallyincreasing doses, or gradually decreasing doses, or further chronicallyor periodically.

Optimal dosing schedules, may be calculated from measurements of drugaccumulation in the body of the patient. Optimal doss can be determinedby dosing methodologies and repetition rates. In general, dosage iscalculated according to body weight, and may be given once or moredaily, weekly, monthly or yearly, or even once every few years. Personsof ordinary skill in the art can readily estimate repetition rates fordosing based on measured residence times and concentrations of thecombined composition of the invention in bodily fluids or tissues.Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the combined composition of the invention is administeredin maintenance doses, once or more daily.

In some embodiments the therapeutically effective amount of trehalose orpharmaceutical formulation comprising same as herein defined isadministered chronically.

The term “chronically” as herein defined refers to a constant regime ofadministration occurring at a predetermined frequency. Thusadministration may be based for example on a daily, weekly or monthlyregime as a single parenteral dose or multiple parenteral, specificallyintravenous doses.

In some embodiments the frequency as herein defined is of between oncedaily to once per month, namely said therapeutically effective amount oftrehalose or pharmaceutical formulation comprising thereof isadministered at a frequency of between once daily to once per month.

In other embodiments the frequency as herein defined is every day(daily), namely seven times per week or six, five, four or three timesper week, twice or once per week, once in every two weeks, once in everythree weeks or once per month.

In specific embodiment the frequency as herein defined is once per week.In other embodiments the therapeutically effective amount of trehaloseor trehalose comprised in said pharmaceutical formulation isadministered at a single injection administration once a week.

In the above and other embodiments the therapeutically effective, amountof trehalose or trehalose comprised in said pharmaceutical formulationis administered once daily at from about 10 mg/kg/day to about 1gram/kg/day of trehalose.

As indicated in the accompanying Examples, in an ongoing clinical studyan aqueous injectable pharmaceutical formulation comprising trehalose isadministered intravenously at a weekly dosing of 8, 15 or 30 gram persubject.

Thus in the above and other embodiments the therapeutically effectiveamount of trehalose or pharmaceutical formulation comprising the same isintravenously administered at a single dose of from about 5 to about 35grams of trehalose, which may be administered once daily, once everyother day, twice a week, once a week, once every two weeks, once everythree weeks or once a month.

In further embodiments the therapeutically effective amount of trehaloseor trehalose comprised in said pharmaceutical formulation isadministered at a single-dose of 5, 8, 15, 30, 40 or 50 grams oftrehalose.

In other embodiments the therapeutically effective amount of trehaloseor a pharmaceutical formulation comprising the same is adapted for aninjectable solution and wherein the rate of administration is such thatthe maximum endotoxin level is less than 5 endotoxin units per kilogramof body weight per hour.

By the term “rate of administration” it is meant the rate of infusion(or dosing rate).

In further embodiments the therapeutically effective amount of trehaloseor a pharmaceutical formulation comprising the same is adapted forintravenous administration and said administration is completed withinless than about 75 to 120 minutes, for example but not limited to within90 minutes. The period of administration will also depend on thevolume/amount of the disclosed injectable trehalose formulation to beadministered.

Thus in some embodiments administration of the therapeutically effectiveamount of trehalose comprised in said pharmaceutical formulation adaptedfor intravenous administration is completed within from about 75 toabout 120 minutes, specifically within less than 90 minutes.

In still further embodiments the administration of trehalose or apharmaceutical formulation comprising the same comprises a dosingregimen of equal doses, or gradually increasing doses, or graduallydecreasing doses of trehalose or pharmaceutical formulation comprisingthe same.

In some embodiments the therapeutically effective amount of trehalose orpharmaceutical formulation comprising thereof is administeredperiodically.

By the term “periodically” it is meant that the administration oftrehalose may be conducted at consecutive chronic administrationregimens as herein defined which may be separated in time by periods of“no treatment” (or “drug holiday”, i.e. when the patient as hereindefined stops taking the active agent for a certain period of time)according to the patient condition.

Determining the efficaciousness of the treatment as herein defined maybe performed by any method known to a person skilled in the art. Forexample, when the disease to be treated is OPMD, efficaciousness of thetreatment may be performed in association with any known method fordiagnosing or treating OPMD. Alleviation of one or more signs orsymptoms of OPMD indicates that the compound confers a clinical benefit.

Thus in some embodiments the disease according to the presentlydisclosed subject matter is OPMD and in such case efficaciousness oftreatment as herein defined may be performed by monitoring patient'sweight; performing a “drinking test”, in which the patient is requestedto drink 80 ml of ice-cold water, and the time which this volume hasbeen fully consumed (in seconds) will be recorded; and by fiberopticendoscopic evaluation of swallowing (FEES), which is a usefulsupplementary tool for studying dysphagia. The FEES procedure involvesintroducing a flexible fiberoptic endoscope transnasally to thepatient's hypopharynx where the clinician can clearly view laryngeal andpharyngeal structures. The patient is then led through various tasks(e.g. food and liquid boluses) to evaluate the sensory and motor statusof the pharyngeal and laryngeal mechanism. Information obtained fromthis examination includes ability to protect the airway, ability tosustain airway protection for a period of several seconds, ability toinitiate a prompt swallow without spillage of material into thehypopharynx, timing and direction of movement of the bolus through thehypopharynx, ability to cleat the bolus during the swallow, presence ofpooling and residue of material in the hypopharynx, timing of bolus flowand airway protection, sensitivity of the pharyngeal/laryngealstructures and the effect of anatomy on the swallow.

Additional tests for evaluating efficaciousness of treatment of OPMDinclude the SWAL-QOL test (a 44 item tool that asks patients to rateSeveral factors about 10 quality-of-life concepts related to swallowingon a 5 point scale) that was developed for measuring objectively apatient's perspective of swallowing and the muscle strength assessment(assessment of weakness of the proximal muscles, including “stair climb”test, “30 second sit-to-stand” test, “30 second weight arm raise” test,etc.).

The term “subject in need thereof” as herein defined is a subjectsuffering from a disease or disorder as herein defined, namely a diseaseassociated with abnormal protein aggregation and/or inclusion bodiesformation in myocytes, neurons and other cells or extracellularcompartments.

In certain embodiments, the present disclosure provides a formulationcomprising trehalose or a physiologically acceptable derivative thereofwherein trehalose (or derivative thereof) or at least a portion of thetrehalose or derivative thereof is formulated for sustained and/orcontrolled release and a portion of the trehalose or derivative thereofis formulated for immediate release, when administered to a subject.

In certain embodiments, effective serum levels of The active ingredienttrehalose or derivative thereof are achieved within from about 10 toabout 20 or 30 or 40 or 50 or 60 minutes following trehaloseadministration. In certain embodiments, effective serum levels of theactive ingredient are achieved within from about 5 to about 20 or 30 or40 or 50 or 60 minutes following trehalose administration. In certainembodiments, effective serum levels of the active ingredient areachieved within from about 20 to about 20 or 30, or 40 or 50 or 60minutes following trehalose administration. In certain embodiments,effective serum levels of the active ingredient are achieved withinabout 5, 10, 15, 20, 30, 40, 50 or 60 minutes followingtrehalose-administration.

The present inventors have developed innovative approaches for theadministration of trehalose based on parenteral routes. These approachesprovide for a rational design of delivery systems (e.g. formulations)with desired properties based on the meticulous selection of thecarrier, e.g. appropriate surfactants/co-surfactants composition ormicro/nano particles (such as liposomes or nano-liposomes) or polymerentrapping the active ingredients, or other additives or excipients, forthe delivery system of interest.

The parenteral ways include subcutaneous and transdermal (diffusionthrough the intact skin) administration. In certain embodiments, theformulations of the invention, are administered by invasive modes oftreatment such as by intravenous, intramuscular and like administration.

Another way of administration can make use of red blood cells loadedwith high quantities of trehalose, which are known in the art. It iscurrently known that loading red blood cells with high amounts oftrehalose enhances their viability. Such red blood cells, highly loadedwith trehalose can serve as a vehicle for intravenous delivery oftrehalose to a subject in need, in accordance with the presentdisclosure.

Administration of trehalose for medical uses requires safe and efficientdelivery systems. The present disclosure provides delivery systems (e.g.formulations for parenteral administration) for safe delivery of avariety of substances due to their special physico-chemical features.The delivery systems significantly enhance efficiency and quality oftrehalose absorption based on its unique physicochemical features, whichenables lower concentrations or amounts of active substance to bedelivered to a subject in a biologically active form. The presentdelivery systems provide for the direct access of the active substanceto the tissues and thus provide immediate or near-immediate effects oftrehalose to the subject in need thereof.

Accordingly, in certain embodiments, the present invention provides apharmaceutical delivery system for the improved administration oftrehalose or physiologically active derivative thereof, comprising asthe active ingredient said trehalose or physiologically activederivative thereof in a suitable carrier for fast restoration of reliefof symptoms of the disease of the treated subject.

In certain embodiments, the drug delivery systems may provide the activesubstance in a controlled release mode. In certain embodiments, the drugdelivery systems of the invention may further comprises at least oneadditional pharmaceutically active agent.

The presently disclosed delivery systems can generally comprise abuffering agent, an agent which adjusts, the osmolality thereof, andoptionally, one or more pharmaceutically acceptable carriers, excipientsand/or additives as known in the art. Supplementary pharmaceuticallyacceptable active ingredients can also be incorporated into thecompositions. The carrier can be solvent or dispersion medium suitablefor parenterally-administrable compositions containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants.

As indicated above, the present trehalose delivery system can beadministered in controlled-, sustained- or delayed-release formulations.Any controlled or sustained release method known to those of ordinaryskill in the art may be used with the formulations and methods of thepresently disclosed subject matter such as those described in Langer1990 [16]. Such method comprises administering a sustained-releasecomposition or a coated implantable medical device so that atherapeutically effective dose of the composition of the invention iscontinuously delivered to a subject of such a method. Sustained releasemay also be achieved using a patch designed and formulated for thepurpose. Controlled or sustained-release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils). Alsocomprehended by the invention are particulate compositions coated withpolymers (e.g., poloxamers or poloxamines). Sustained release formulaeor devices, or any topical formulations, may additionally containcompositions to stabilize the composition or permeate physiologicalbarrier such as skin or mucous membrane. Exemplary additional componentsmay include any physiologically acceptable detergent, or solvent suchas, for example, dimethylsulfoxide (DMSO).

In certain embodiments, the trehalose in the present compositions can beformulated for sustained or controlled release over a period of at least0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. In certainembodiments, the trehalose in the present compositions can be formulatedfor sustained or controlled release over a period of about 0.5, 1, 2, 3,4, 5, 7, 8, 9, 10, 11 or 12 hours. In certain embodiments, the trehalosein the present compositions can be formulated for sustained orcontrolled release over a period of between about 0.5 or 1 or 2 or 3 or4 hours and about 5, 6, 7, 8, 9, 10, 11 or 12 hours. In certainembodiments, the trehalose in the present compositions can be formulatedfor sustained or controlled release over a period of between about 5 or6 or 7 or 8 hours and about 9, 10, 11 or 12 hours.

In certain embodiments the trehalose in the present compositions can bein immediate, fast of burst release form.

In certain embodiments, the trehalose in the present compositions can beformulated to release up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5 or 100% of the total trehalosein about 0.5, 1, 2, 3, 4, 5, 6, 7 or 8 hours. In certain embodiments,the trehalose in the present compositions can be formulated to releasenot less than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 99, 99.5 or 100% of the total trehalose in about 0.5, 1,2, 3, 4, 5, 6, 7 or 8 hours.

In certain embodiments, the trehalose in the present compositions can bein a combination of sustained or slow release and immediate or fast orburst release forms. In certain embodiments, the relative proportion ofsustained or slow release trehalose to immediate or fast releasetrehalose is, e.g., 1 to 99, 5 to 95, 10 to 90, 15 to 85; 20 to 80, 25to 75, 30 to 70, 35 to 65, 40 to 60, 45 to 55, 50 to 50, 55 to 45; 60 to40, 65 to 35, 70 to 30, 75 to 25, 80 to 20, 85 to 15, 90 to 10, 95 to 5,or 99 to 1.

In certain embodiments, a polymeric material is used to sustain orcontrol release of trehalose. In certain embodiments, the type ofpolymeric material and the amount of which is used, have a stronginfluence on the rate of release of trehalose from the presentcompositions and delivery systems. Examples of polymers include bothhydrophobic and hydrophilic polymers. Examples of hydrophobic polymersinclude, but are not limited to, ethyl cellulose and other cellulosederivatives, fats such as glycerol palmito-stearate, beeswax, glycowax,castorwax, carnaubawax, glycerol monostearate or stearyl alcohol,hydrophobic polyacrylamide derivatives and hydrophobic methacrylic acidderivatives, as well as mixtures of these polymers. Hydrophilic polymersinclude, but are not limited to, hydrophilic cellulose derivatives suchas methyl cellulose, hydroxypropylmethyl cellulose,hydroxyethylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose,sodium carboxymethylcellulose and hydroxyethyl methylcellulose polyvinylalcohol, polyethylene, polypropylene, polystyrene, polyacrylamide,ethylene vinyl acetate copolymer, polyacrylate, polyurethane,polyvinylpyrrolidone, polymethylmethacrylate, polyvinyl acetate,polyhydroxyethyl methacrylate, as well as mixtures of these polymers.Furthermore, any mixture of one or more hydrophobic polymer and one ormore hydrophilic polymer could optionally be used.

The trehalose contained in the present compositions and delivery systemscan be entrapped in liposomes, micro- and nano-particles.

In certain embodiments, a polymeric material to be used in the presentcompositions and delivery systems is microcrystalline cellulose such as“Avicel PH 101” manufactured by FMC BioPolymer's. Alternatively, apolymeric material to be used in the present compositions and deliverysystems is hydroxypropyl methylcellulose such as “Metholose” produced byShin-Etsu Chemical Co. In certain embodiments, a polymeric material tobe used in the present compositions and delivery systems is ethylcellulose such as “Ethocel™” manufactured by The Dow Chemical Company.In certain embodiments, a polymeric material to be used in the presentcompositions and delivery systems is an acrylic polymer such as“Eudragit RS™” produced by Rohm GmbH. In certain embodiments, apolymeric material to be used in the present compositions and deliverysystems is a colloidal silicone dioxide such as “Acrosil™” manufacturedby Degussa. In certain embodiments, a polymeric material to be used inthe present compositions and delivery systems is a Poly (Vinyl Acetate)such as “Kollicoat SR” manufactured by BASF. In certain embodiments, apolymeric material to be used in the present compositions and deliverysystems is an ethyl acetate and vinyl acetate solution such as“Duro-Tak” manufactured by Delasco Dermatologic Lab & Supply, Inc.

In certain embodiments, delivery systems of the invention comprisedelivery devices. In certain embodiments, the compositions of theinvention are delivered by ail osmotic process at a controlled rate suchas by an osmotic pump. The system may be constructed by coating anosmotically active agent with a rate controlling semipermeable membrane.This membrane may contain an orifice of critical size through whichagent is delivered. The dosage form after coming into contact withaqueous fluids, imbibes water at a rate determined by the fluidpermeability of the membrane and osmotic pressure of the coreformulation. This osmotic imbibitions of water result in formation of asaturated solution of active material with in the core, which isdispensed at controlled rate from the delivery orifice in the membrane.

In certain embodiments, the compositions of the invention are deliveredusing biodegradable microparticles. In certain embodiment, the system toprepare microparticles consists of an organic phase comprised of avolatile solvent with dissolved polymer and the material to beencapsulated, emulsified in an aqueous phase. In certain embodiments,the biodegradable polymers that can be used for the microparticlematrix, comprises polylactic acid (PLA) or the copolymer of lactic andglycolic acid (PLAGA). The PLAGA polymer degrades hydrolytically overtime to its monomeric components, which are easily removed from the bodythrough natural life processes.

The preparation may also contain an absorption enhancer and otheroptional components. Examples of absorption enhancers include, but arenot limited to, are cyclodextrins, phospholipids, chitosan, DMSO, Tween,Brij, glycocholate, saponin, fusidate and energy based enhancingabsorption equipment.

Optional components present in the dosage forms include, but are notlimited to, diluents, binders, lubricants, surfactants, coloring agents,flavors, buffering agents, preservatives, stabilizing agents and thelike.

Diluents, also termed “fillers” include, for example, dicalciumphosphate dihydrate, calcium sulfate, lactose, cellulose, kaolin;mannitol, sodium chloride, dry starch, hydrolyzed starches, silicondioxide, colloidal silica, titanium oxide, alumina, talc,microcrystalline cellulose, and powdered sugar. For administration inliquid form, the diluents include, for example, ethanol, sorbitol,glycerol, water and the like.

Binders are used to impart cohesive qualities to the formulation.Suitable binder materials include, but are not limited to, starch(including corn starch and pregelatinzed starch), gelatin, sugars(including sucrose, glucose, dextrose, lactose and sorbitol),polyethylene glycol, waxes, natural and synthetic gums, e.g., acacia,tragacanth, sodium alginate, celluloses, and Veegum, and syntheticpolymers such as polymethacrylates and polyvinylpyrrolidone.

Lubricants are used to facilitate manufacture; examples of suitablelubricants include, for example, magnesium stearate, calcium stearate,stearic acid, glyceryl behenate, and polyethylene glycol.

Surfactants may be anionic, cationic, amphoteric or nonionic surfaceactive agents, with anionic surfactants preferred. Suitable anionicsurfactants include, but are not limited to, those containingcarboxylate, sulfonate and sulfate ions, associated with cations such assodium, potassium and ammonium ions. Particularly preferred surfactantsinclude, but are not limited to: long alkyl chain sulfonates and alkylaryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodiumsulfosuccinates, such as sodium bis-(2-ethylhexyl)-sulfosuccinate; andalkyl sulfates such as sodium lauryl sulfate.

Stabilizing agents such as antioxidants, include, but are not limitedto, propyl gallate, sodium ascorbate, citric acid, calciummetabisulphite, hydroquinone, and 7-hydroxycoumarin.

If desired, the present compositions may also contain minor amounts ofnontoxic auxiliary substances such as wetting or emulsifying agents,preservatives, and the like.

As mentioned, any of the compositions of the invention may be used aloneor in combination with one or more additional therapeutic agents, forthe treatment of the disease from which the treated subject suffers. Theamount of both the compound and the additional therapeutic agent thatmay be combined with the carrier materials to produce a composition ordelivery system will vary depending upon the host treated and theparticular mode of administration. In some embodiments, the compositionsof this invention should be formulated so that a dosage of between0.01-1 g/kg body weight/day of trehalose can be administered. The doseof the trehalose depends on the condition and the illness of thepatient, and the desired daily dose. In human therapy, the daily dosecan be 10-3000 mg, for example, 10, 15, 20, 30, 40, 50, 75, 100, 200,300, 400, 500, 600 700, 800, 900 or 1000 mg. These amounts areadministered in doses which can be divided into 2-3 smaller doses foreach day.

In certain embodiments, the active ingredients in the presentcompositions and delivery systems can act synergistically in combinationwith each other and can further act synergistically in the presence ofan additional therapeutic agent. Therefore, the amount of compound(s)and additional therapeutic agent(s) in such compositions will be lessthan that required in a monotherapy utilizing only that therapeuticagent.

Toxicity and therapeutic efficacy of the formulations described hereincan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., procedures used for determining themaximum tolerated dose (MTD), the ED₅₀, which is the effective close toachieve 50% of maximal response, and the therapeutic index (TI), whichis the ratio of the MTD to the ED₅₀. Obviously, formulations with highTIs are the most preferred formulations herein, and preferred dosageregimens are those that maintain plasma levels of the trehalose at orabove a minimum concentration to maintain the desired therapeuticeffect. Dosage will, of course, also depend on a number factors, thesite of intended delivery, the route of administration, and otherpertinent factors known to the prescribing physician.

As indicated above, trehalose or any of the formulations comprisingthereof as herein defined may be used alone or in combination with oneor more additional therapeutic agents for the treatment of the diseasesfrom which the treated subjects suffer. The amount of both the compoundand the additional therapeutic agent that may be combined with trehaloseor any of the formulations comprising thereof as herein defined willvary upon the subject treated and the particular diseases and mode ofadministration.

In certain embodiments, the active ingredients in the presentcompositions and delivery systems can act synergistically in combinationwith each other and can further act synergistically in the presence ofan additional therapeutic agent. Therefore, the amount of compound(s)and additional therapeutic agent(s) in such compositions will be lessthan that required in a monotherapy utilizing only that therapeuticagent.

In another one of its aspects the presently disclosed subject matterprovides Trehalose or a pharmaceutical formulation comprising same, foruse in a method for treating or alleviating a disease associated withabnormal protein aggregation and/or inclusion bodies formation inmyocytes, neurons and other cells or extracellular compartments or atleast one symptom associated therewith, in a human subject in needthereof, said method comprising parenterally administering to saidsubject a therapeutically effective amount of trehalose or apharmaceutical formulation comprising the same.

In yet another one of its aspects the presently disclosed subject matterprovides an aqueous pharmaceutical formulation comprising atherapeutically effective amount of trehalose as a sole activeingredient, wherein the formulation has a pH about 4.5 to 7.0 andcontains less than 0.74 endotoxin units per ml and wherein saidpharmaceutical formulation is adapted for parenteral administration.

In some embodiments the aqueous pharmaceutical formulation as hereindefined may be adapted for intravenous, intramuscular or intraperitonealadministration.

In other embodiments the aqueous pharmaceutical formulation as hereindefined is adapted for intravenous, intramuscular or intraperitonealadministration.

As indicated above the aqueous pharmaceutical formulation as hereindefined optionally further comprises at least one pharmaceuticallyacceptable additive, carrier, excipient or diluents.

In further embodiments the aqueous pharmaceutical formulation as hereindefined is wherein the concentration of the trehalose is between about0:1% (w/v) to about 50% (w/v). In further specific embodiments theaqueous pharmaceutical formulation as herein defined is wherein theconcentration of the trehalose is about 10% (w/v).

In some embodiments the aqueous pharmaceutical formulation as hereindefined is wherein the formulation has an osmolality of about 280-330mOsm/kg.

In other embodiments the aqueous pharmaceutical formulation as hereindefined is wherein the formulation is administered at a frequencybetween once daily to once per month.

The aqueous pharmaceutical formulation, as herein defined may be wheresaid therapeutically effective amount of trehalose is from about 1 gramto about 100 gram for each daily injection and no more than about 1gram/kg body weight of said subject per day.

In other embodiments the aqueous pharmaceutical formulation as hereindefined is wherein the formulation is administered once daily at fromabout 10 mg/kg/day to about 1 gram/kg/day of trehalose.

In still further embodiments the aqueous pharmaceutical formulation asherein defined is wherein the formulation is administered at a frequencyof between once daily to once per month at a dose of about 5 to about 35grams of trehalose.

In further specific embodiments the aqueous pharmaceutical formulationas herein defined is for administration once daily, once every otherday, twice a week, once a week, once every two weeks, once every threeweeks or once a month.

In still further specific embodiments the aqueous pharmaceuticalformulation as herein defined is wherein the dose is 5, 8, 15, 30, 40 or50 grams.

In yet further embodiments the aqueous pharmaceutical formulation asherein defined is wherein the rate of administration is such that themaximum endotoxin level is less than 5 endotoxin units per kilogram ofbody weight per hour.

The aqueous pharmaceutical formulation of the presently disclosedsubject matter may be where the formulation is adapted for intravenousadministration and wherein said administration is completed within fromabout 75 to about 120 minutes, specifically within less than 90 minutes.

In some embodiments the aqueous pharmaceutical formulation as hereindefined is for treating a disease associated with abnormal proteinaggregation and/or inclusion bodies formation in myocytes, neurons andother cells or extracellular compartments or for alleviating a sign orsymptom associated therewith in a human subject in need thereof.

The presently disclosed subject matter further provides a kitcomprising:

-   -   (a) pharmaceutically acceptable trehalose or active derivative        thereof;    -   (b) at least one pharmaceutically acceptable additive, carrier,        excipient and diluent;    -   (c) means for preparing an injectable aqueous solution of the        trehalose by mixing said trehalose with at least one of said        additive, carrier, excipient and diluent;    -   (d) means for parenterally administering said injectable        solution to a patient in need;    -   (d) instructions for use.

Injectable aqueous solution of the trehalose may be prepared by anymethod well known in the art, for example as recited herein in theaccompanying Examples. Parenterally administering the injectablesolution as herein defined well known in the art of the skilledphysician.

The term “about” as used herein indicates values that may deviate up to1%, more specifically 5%, more specifically 10%, more specifically 15%,and in some cases up to 20% higher or lower than the value referred to,the deviation range including integer values, and, if applicable,non-integer values as well, constituting a continuous range.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

Various embodiments and aspects Of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, methods steps, and compositionsdisclosed herein as such methods steps and compositions may varysomewhat. It is also to be understood that the terminology used hereinis used for the purpose of describing particular embodiments only andnot intended to be limiting since the scope of the present inventionwill be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise.

The following examples are representative, of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the spirit and intendedscope of the invention.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe claimed invention in any way.

Standard molecular biology protocols known in the art not specificallydescribed herein are generally followed essentially as in Sambrook &Russell, 2001.

Standard medicinal chemistry methods known in the art not specificallydescribed herein are generally followed essentially in the series“Comprehensive Medicinal Chemistry” by various authors and editors,published by Pergamon Press.

Example 1 Trehalose

Trehalose, dihydrate was obtained from two different commercial sources(Hayashibara and Pfanstiehl). The samples were kept at room temperatureuntil analysis, and were analyzed by accepted methods to verifyspecifics. The results are presented in Table 1 below.

TABLE 1 Analysis of trehalose dihydrate Test Required SpecificationsSample Pfanstiehl Assay 97.0-102.0% w/w on 99.4% (conforms) anhydrousbasis Related Maltotriose Conforms substances ≤0.5% w/w Any peak elutingbefore Conforms trehalose ≤0.5% w/w Glucose ≤0.5% w/w Conforms Any peakeluting after Conforms trehalose ≤0.5% w/w

As shown in Table 1 above, the tested trehalose dihydrate preparationsconform with the accepted practice concerning use of trehalose inpharmaceutical compositions, namely comprise 97.0-102.0% w/w activeingredient (trehalose), with any peak eluting before trehalose at aconcentration of less than 0.5% w/w, glucose at less than 0.5% w/w andwith any peak eluting after trehalose at less than 0.5% w/w.

The trehalose used was commercially available trehalose dihydrate,comprising 97.0-102.0% w/w active ingredient (trehalose), with glucoseat less than 0.5% w/w and other contaminants at less than 1%.

Example 2 Preparation of Trehalose Solution for IV Injection

A formulation comprising trehalose was prepared under sterile conditionsby dissolving trehalose dihydrate in water and the resulting clear andcolorless liquid was analyzed to identify any impurities orcontaminants.

TABLE 2 Analysis of trehalose formulation Test Specification ResultAppearance of container Clear glass 30R vial with grey Clear glass 30Rvial with grey ATP007 rubber stopper, aluminium seal and rubber stopper,aluminium seal white flip off lid and white flip off lid. Appearance ofcontents Clear colourless liquid essentially Clear colourless liquidATP007 free from visible particulate matter essentially free fromvisible particulate matter P537 Identity ATP1323 Retention time of theP537 peak ± 5% Retention time of the P537 of standard peak is within ±5% of the P537 standard peak P537 Content Assay 90.0% to 110.0% labelclaim 99.8% ATP1323 P537 Related Substances Report individual impuritiesMaltotriose: None detected (% label claim) ATP1323 ≥0.05% label claimUnknown RRT0.90: 0.1% Report total impurities Glucose: None detectedMaltotriose and other Total Impurities: 0.1%. polysaccharides elutingbefore P537: ≤0.5% Glucose and peaks eluting after P537 ≤0.5% Totalimpurities 2.0% pH 4.5 to 7.0 6.2 ATP164 Osmolality ATP841 280-330mOsm/kg 289 mOsm/kg Particulate matter Particulates ≥10 μm: NMT 6000 ≥10μm: 7 USP <788>, Ph. Eur Particulates ≥25 μm: NMT 600 ≥25 μm: 0 2.9.19Extractable Volume Not less than 30 ml 32 ml USP <I>, Ph. Eur 2.9.17Endotoxins <0.24 EU/ml Point 1 tray 1: <0.1 EU/ml USP <85>, Ph. Eur2.6.14 Point 4 tray 31: <0.1 EU/ml Point 8 tray 72: <0.1 EU/ml SterilityComplies No growth USP <71>, Ph. Eur 2.6.1

The formulation was sterile and comprised 10% trehalose, with totalimpurities at a concentration of 0.1%, pH of 6.2 and an osmolality of289 mOsm/kg and endotoxins at less than 0.1. EU/ml.

Example 3 Preclinical Pharmacokinetic Studies

The plasma and muscle concentrations of trehalose in male Sprague-Dawley(SD) rats was determined after intravenous bolus (IV) and oral gavage(PO) administration.

All applicable portions of the study confirmed to the followingregulations, and guidelines regarding animal care and welfare: AAALACInternational and NIH guidelines as reported in the “Guide for the Careand Use of Laboratory Animals;” National. Research Council ILAR, Revised1996.

The study included 42 SD rats (male, 250 to 350 grams in weight, theShanghai SLAC Laboratory Animal Co. Ltd.). Animals were administeredwith a volume of 5 ml/kg trehalose formulation (trehalose dihydrate insterilized, water at 200 mg/mL) to achieve a nominal dose of 1 gr/kg,intravenously or orally.

Blood samples were collected after each dose administration andprocessed for plasma. Muscle samples (hind leg muscle) were collectedand homogenized. The concentrations of trehalose in plasma and musclehomogenate samples were analyzed by qualified bioanalytical LC/MS/MSmethods.

Pharmacokinetics Data Analysis

Plasma concentration data of trehalose was subjected to a noncompartmental pharmacokinetic analysis using WinNonlin software program(version 6.3, Pharsight, Mountain View, Calif.). Zero-time interceptconcentration (C0), volume of distribution (Vdss), Clearance (Cl), peakplasma concentrations (Cmax) and the corresponding peak times (Tmax),terminal half-life (T½), mean residence time (MRT) from time zero to thelast time point (MRT0-last), MRT from time zero to infinity (MRT0-inf),the area under the plasma concentration-time curve (AUC) from time zeroto the last time point (AUC0-last) and AUC from time zero extrapolatedto infinity (AUC0-inf) were calculated using the linear/log trapezoidalrule. Nominal sampling times were used, to calculate all pharmacokineticparameters since there was not any deviation larger than 5% between theactual and nominal sampling times.

The values of muscle to plasma concentration and AUC ratio (M/P ratio)were both calculated.

Trehalose Concentration in Plasma and Muscle

Pharmacokinetic parameters of trehalose in the plasma and musclefollowing single intravenous or oral administration of trehalosedihydrate solution (200 mg trehalose dihydrate per 1 mL sterilizedwater) at 1000 mg/kg to male SD rats are presented in Table 3 below.

Individual and mean plasma concentrations of trehalose followingintravenous or oral administration of trehalose dihydrate solution (200mg trehalose dihydrate per 1 mL sterilized water) at 1000 mg/kg to maleSD rats are presented in Table 3 and shown graphically in FIG. 1.

Individual and mean muscle concentrations of trehalose following singleintravenous or oral administration of trehalose dihydrate solution (209mg trehalose dihydrate per 1 mL sterilized water) at 1000 mg/kg to maleSD rats are presented in Table 3 as well. Plasma and muscleconcentrations comparison for trehalose following single intravenous ororal administration of trehalose dihydrate at 1000 mg/kg to male SD ratsare nd shown graphically in FIGS. 2 to 3.

Following a single intravenous dose of trehalose solution (200 mgtrehalose dihydrate per 1 mL sterilized water) at 1000 mg/kg to malefasted SD rats in tested groups 1 to 5, trehalose showed a totalclearance (Cl) of 17.2 mL/min/kg (approximately 31.3% of rat liver bloodflow (=55 mL/min/kg)), with the averaged elimination half life (T_(1/2))of 2.07 hours. The C₀ was 1,370,000 ng/mL.

The volume distribution (V_(dss)) was at 0.685 L/kg. The mean plasmaexposure AUC_(0-last) (48 hr) was 778,000, ng·hr/mL.

With an oral administration of trehalose dihydrate solution (200 mgtrehalose dihydrate per 1 mL sterilized water) to male SD rats in testedgroups 6 to 10, trehalose maximum plasma concentration (C_(max)=4,280,ng/mL) was attained at 0.5 hour post dose (T_(max)). The AUC_(0-last) (3hr) was 4,520 ng/mL·hr. The absolute bioavailability of trehalose wasestimated tote as low as 0.601%.

The pharmacokinetic properties of trehalose demonstrated a rapidabsorption with a time to reach peak plasma concentrations, but theabsolute oral bioavailability was very low, which noted that thecompound may undergo a significant presystemic-metabolism.

Following a single intravenous dose of trehalose dihydrate solution (200mg trehalose dihydrate per 1 mL sterilized water) at 1000 mg/kg to maleSD rats in tested groups 1 to 5, the C_(max) of trehalose in muscle was3730 ng/mL, which was observed at 8 hours (T_(max)) post dose. Themuscle exposure AUC_(0-last) (48 hr) was 107,000 ng·hr/mL with theelimination half life 33.8 hours. The PK parameters of muscle samples indrug treated oral group (Groups 6 to 10) could not be calculated becausethey were below LLOQ

The mean ratios of muscle trehalose concentration to plasmaconcentration ranged froth 2.88 to 3.76 in male SD rats followingintravenous administration. Muscle to plasma concentration ratios fortrehalose upon oral administration were below LLOQ and not calculable.

CONCLUSIONS

Following intravenous or oral administrations of trehalose dihydratesolution in sterilized water at 1000 mg/kg to male SD rats, trehalose inplasma and muscle tissue were determined. Plasma glucose was alsomonitored for each sample from study animals of drug treated groups. Thefollowing conclusions can be made:

First, following IV administration, the total clearance (Cl) oftrehalose was 17.2 mL/min/kg, accounting for approximately 3.1.3% ofliver blood flow, a moderate value of hepatic extraction ratio. TheV_(dss) and T_(1/2) were 0.685 L/kg, and 2.07 hours respectively. Themean plasma exposure AUC_(0-last) was 778,000 ng·hr/mL.

Following oral administration, trehalose demonstrated a rapid absorptionwith T_(max) observed at 0.50 hours post dose, but the absolute oralbioavailability was as low as 0.601%, suggesting presystemic metabolismmay play an important role. T_(1/2) of trehalose was markedly shortenedin oral administration rats in comparison to the intravenous group.

Following IV administration, the mean ratios of muscle trehaloseconcentration to plasma concentration, ranged from 2.88 to 3.76 in maleSD rats.

Finally, it was observed that trehalose dihydrate was well tolerated bythe rats at the given dosage.

TABLE 3 Pharmacokinetic parameters Matrix Plasma Muscle Group ID IV POIV PO C₀ (ng/mL) 1370000 — — ND C_(max) (ng/mL or ng/g) — 4280 3730 NDT_(max) (h) — 0.500 8.00 ND T_(1/2) (h) 2.07 0.740 33.8 ND CI(mL/min/kg) 17.2 — — ND V_(dss) (L/kg) 0.685 — — ND AUC_(0-last) (ng ·h/mL or 778000 4520 107000 ND ng · h/g) AUC_(0-inf) (ng · h/mL or 7810004870 183000 ND ng · h/g) MRT_(0-last) (h) 0.618 1.04 21.1 ND MRT_(0-inf)(h) 0.666 1.26 52.6 ND AUC_(0-inf)/AUC_(0-last) 100 108 171 ND (%)^(c)Bioavailability (%) — 0.601 — — ^(d)AUC ratio — — 0.234 NDAbbreviations: ND = Not determined; ^(c)Bioavailability (%) wascalculated with mean AUC_(0-inf) and norminal dose; ^(d)AUC Ratio =Muscle AUC_(0-inf)/Plasma AUC_(0-inf); AUC_((0-inf)) >120% ofAUC_((0-last)).

Thus, as demonstrated in Table 3, the trehalose in the IV administeredformulation showed T_(1/2) of 2.07 hour in plasmas, over two-fold higherthan the plasma T_(1/2) obtained for trehalose in the orallyadministered formulation (0.740 hours). In addition, the muscle T_(1/2)obtained for the trehalose in the IV administered formulation was 33.8hours. The AUC values obtained for plasma and muscle when theformulation was administered IV were also significantly higher than therespective AUC values of formulation administered orally.

In addition, as demonstrated in FIG. 1, the mean plasma concentration ofthe trehalose in the IV administered formulation is higher than the meanplasma concentration of the trehalose in the orally administeredformulation at each of the tested, time points.

Interestingly, FIG. 2, which demonstrates plasma versus muscleconcentrations of trehalose for a trehalose formulation administeredintravenously, shows, that muscle concentrations am higher than plasmaconcentrations of trehalose. Plasma and muscle concentrations oftrehalose were undetectable for a trehalose formulation administeredorally.

Example 4 Determination of Endotoxin Level

It is accepted that the maximal allowed level of endotoxin informulations administered intravenously is 5 endotoxin units (EU) per kgbody Mass per hour (5 EU/kg/hr). In order to determine the theoreticalmaximum endotoxin level IV per kg body mass/hour (K) in trehaloseformulation (solution of trehalose dihydrate in sterilized water), thefollowing calculations were made:

TABLE 4 Calculation of maximal endotoxin levels in trehaloseformulations 30 gr trehalose Endotoxin contribution 15 gr trehaloseformulation formulation 2.4 EU/gr (trehalose) 36 72 0.5 EU/ml (solvent)75 (in 150 ml) 150 (in 300 ml) Total EU in formulation 111 222 Assuming75 min infusion 88.8 EU/hr 177.6 EU/hr K for 60 kg body weight 1.5 3.0 Kfor 50 kg body weight 1.8 3.6 K for 40 kg body weight 2.2 4.4

As indicated in Table 4 above, endotoxin level per ml in trehaloseformulations prepared with standard solvents (e.g. water, saline, etc.),is 0.74 EU/ml. Assuming a moderate infusion rate of 75 minutes, for abody weight of 60, 50 and 40 kg the endotoxin level in trehalose 10%(w/v) formulations is 1.5, 1.8 and 2.2 EU/kg/hr, respectively, for aformulation comprising 15 gr trehalose and 3.0, 3.6 and 4.4 EU/kg/hr,respectively, for a formulation comprising 30 gr trehalose.

Accordingly, under the maximum rate planned, the endotoxin level for abody weight of 60, 50 and 40 kg will be 2, 2.4 and 3 EU/kg/hr,respectively, for a formulation comprising 15 gr trehalose (in 150 mlsolvent) and 4, 4.8 and 6 EU/kg/hr, respectively, for a formulationcomprising 30 gr trehalose (in 300 ml solvent).

Example 5 Safety Studies

The safety and tolerability of trehalose has been extensivelyinvestigated, as detailed below. Trehalose median lethal dose (LD₅₀) wasexamined in mice, rats and dogs. Neither species showed any signs oftoxicity and no deaths occurred after oral and intravenousadministration. The results are summarized in Table 5 below:

TABLE 5 LD50 of trehalose in animals SPECIES ROUTE LD50 (mg/kg bw) MouseOral >5000 Mouse Intravenous >1000 Rat Oral >16000 Rat Oral >5000 RatIntravenous >1000 Dog Oral >5000 Dog Intravenous >1000

In addition, trehalose is recognized as a safe food ingredient as wellas a GRAS material used in the pharmaceutical industry as an excipientfor oral, intraocular and IV drug formulations. In several studies,healthy volunteers were given oral doses of trehalose ranging from 10 to60 gr. Apart from mild abdominal symptoms (e.g. flatulence, distension,borborygmus and occasional diarrhea) no other safety issues werereported [18].

Trehalose has been used as a protein stabilizer in several commerciallyavailable protein drugs for over a decade and its safety has repeatedlybeen established in patient populations at advanced stages of malignantdiseases, hemophilia and related clotting disorders. These drugs areapproved for use for, several years, and are sometimes given to patientsas frequently as every 8 hours through 2-3 weeks intervals.

Example 6 Treatment of OPMD Patients with Trehalose

The potential benefit of trehalose in ameliorating the symptoms of OPMDor slowing the deterioration of OPMD patients, is currently examined ina clinical study in human patients.

The study is a randomized, double-blind, dose escalation andparallel-group dose-controlled study of treatment of patients withOculopharyngeal Muscular Dystrophy (OPMD) IV with trehalose aqueousinjectable solution is carried out in three medical centers.

Exploratory Phase Screening Period (Week-4/Day-28 to Week 0/Day 0)

Screening assessments are conducted over two visits within 28 days priorto the start of therapy, as specified in the Schedule of Assessments.

Treatment Period 1 (Week 1 to Week 24)

All eligible patients receive study treatment once a week.

Initially, all eligible patients receive intravenously one dose oftrehalose (8 g) in a 10% (w/v) injectable aqueous solution per week (80ml solution). This is followed by intravenous administration Of afurther single trehalose dose (15 g) in a 10% (W/v) injectable aqueoussolution during the following week. Following determination of safety,at the next visit on subsequent week, patients are randomized at a 1:1ratio (double-blind) to receive either 15 g or 30 g of trehalose (in anaqueous 10% (w/v) solution) for 24 weeks. The first 4 infusions are doneat the clinic under professional direction. Patients return to theclinic once a month for drug infusion and study assessments, asindicated in the Schedule of Procedures; all other weekly infusions Maybe done in the patient's home or in the clinic.

Pivotal Phase Treatment Period 2 (Week 25 to Week 72)

Patients continue weekly IV infusions of trehalose aqueous injectablesolution.

Follow-Up Period (4 Weeks Post-Dose)

Patients are seen at a post-treatment follow-up visit, 4 weeks after thefinal dose of 24^(th) week.

Study Population

Up to 15 adult patients with OPMD are enrolled into the study at each ofthe three study centers. A minimum of 42 patients are enrolled in total.A control group receiving no treatment may be added.

Inclusion Criteria

-   1. Males and females-   2. 18-80 years (inclusive) of age-   3. Genetically diagnosed with OPMD-   4. Moderate dysphagia (abnormal drinking test at screening and on    the first dosing day, before drug administration)-   5. Patients must be ambulatory; and capable of performing the muscle    functional and strength assessments-   6. Patients who provide written informed consent to participate in    the study-   7. Body Mass Index (BMI) <30 kg/m²-   8. Female patients of child-bearing potential must have a negative    serum pregnancy test at screening-   9. Male and females must agree to use acceptable birth control

Exclusion Criteria

-   1. Diabetes mellitus Type 1 or 2-   2. Other major diseases, e.g. renal failure (creatinine clearance    <60 ml/min), liver failure and chronic liver diseases (e.g.    hepatitis B or C), HIV carriers, tuberculosis, SLE, rheumatoid    polyarthritis, sarcoidosis, collagenosis.-   3. Uncontrolled heart disease, e.g., CHF.-   4. Other neuromuscular diseases.-   5. Other disorders associated with esophageal dysphagia: e.g.    gastroesophageal reflux (GERD), esophageal stricture due to    mechanical or chemical trauma, infection (e.g. esophageal    moniliasis), drug-induced dysphagia (e.g. bisphosphonates),    esophageal rings and webs, spastic motility disorders of the    esophagus.-   6. History of malignancy.-   7. History of neck irradiation.-   8. Pregnant or currently lactating women.-   9. Obesity (BMI≥30) and associated morbidity.-   10. Prior pharyngeal myotomy.-   11. Weight loss of more than 10% in the last 12 months.-   12. Known hypersensitivity to any ingredients in the injection.

Investigational Product Route and Dosage Form

A 10% (w/v) IV solution of trehalose is administered in a singleinjection, once a week for 72 weeks. The solution is delivered overapproximately from about 70 to about 120 minutes.

Initial dose per injection in Week 1 is 8 g trehalose, Second week doseis 15 g, and dose at weeks 3 to week 72 is 15 or 30 g.

Safety and Tolerability

The primary safety endpoint is the frequency, severity, and duration ofadverse events (AEs), including clinically significant laboratoryabnormalities after administration of the trehalose injectable solution.

Safety is evaluated on the basis of AEs and concomitant medications;physical examination; vital signs (prior to, every 30 minutes during,and 30 minutes following administration). Test include complete bloodcount (CBC) with differential, electrolytes (Na, K, Cl), BUN,creatinine, glucose, liver function tests (ALT, AST, total bilirubin,direct bilirubin, alkaline phosphatase, and serum albumin), and dipstickurinalysis.

Efficacy Outcomes/Disease Markers Evaluation

The following disease markers are assessed at specified times:

-   -   Penetration Aspiration Score (using Videofluoroscopy)    -   SWAL-QOL    -   Muscle timed functional and strength assessments

Changes compared to baseline will be measured for each patient, and thetotal change in scores for the treatment groups in each pre-determinedefficacy endpoint is statistically analyzed.

Additional assessments may be of weight and drinking.

Percutaneous Core Needle Biopsy (PCNB) is performed to obtain musclefiber for histology.

Pharmacokinetics

The pharmacokinetic of trehalose are assessed in patients at the dose of15 g or 30 g trehalose. Trehalose blood concentration is measuredpre-dose (up to 60 minutes before drug administration); and every 30minutes after dosing is initiated, for 5 hours or until glucose levelsreturn to normal, whichever occurs first.

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The invention claimed is:
 1. A method for ameliorating a symptom in asubject wherein the subject has a disorder selected from:spinocerebellar ataxia (SCA) associated with a polyglutamine repeatmutation, spinal and bulbar muscular atrophy (SBMA),dentatomral-pailidoluyssan atrophy (DRPLA), Pick's disease, corticobasaldegeneration (CBD), progressive supranuclear palsy (PSP), andfrontotemporal dementia; wherein the method comprises parenterallyadministering to said subject a pharmaceutical formulation comprisingtrehalose as the sole active ingredient; wherein the concentration oftrehalose in said formulation is between about 0.1% (w/v) to about 50%(w/v); and wherein the administration is completed within less than 120minutes.
 2. The method of claim 1, wherein said pharmaceuticalformulation is an injectable solution for parenteral administration. 3.The method of claim 1, wherein said pharmaceutical formulation furthercomprises at least one pharmaceutically acceptable additive, carrier,excipient, or diluent.
 4. The method of claim 1, wherein said parenteraladministration is any one of intravenous, intramuscular, orintraperitoneal administration.
 5. The method of claim 1, wherein thepharmaceutical formulation has a pH about 4.5 to 7.0.
 6. The method ofclaim 1, wherein the pharmaceutical formulation contains less than 0.75endotoxin units per mL.
 7. The method of claim 1, wherein thepharmaceutical formulation is administered to the subject at a peradministration dose of about 10 to about 1000 mg/kg subject body weight.8. The method of claim 7, wherein the pharmaceutical formulation isadministered to the subject at a per administration dose of about 30 toabout 100 mg/kg subject body weight.
 9. The method of claim 7, whereinthe formulation is administered to the subject at a per administrationdose of about 100 to about 300 mg/kg subject body weight.
 10. The methodof claim 7, wherein the pharmaceutical formulation is administered tothe subject at a per administration dose of about 100 to about 150 mg/kgsubject body weight.
 11. The method of claim 7, wherein thepharmaceutical formulation is administered to the subject at a peradministration dose of about 200 to about 600 mg/kg subject body weight.12. The method of claim 1, wherein the pharmaceutical formulationfurther comprises a trehalase inhibitor.
 13. The method of claim 1,wherein the pharmaceutical formulation is administered to the subjectdaily.
 14. The method of claim 1, wherein the pharmaceutical formulationis administered to the subject twice per day.